[CANCER RESEARCH 50, 4167-4172, July 1, 1990]
Rhodamine 123 Phototoxicity in Laser-irradiated
MGH-U1 Human Carcinoma
Cells Studied in Vitro by Electron Microscopy and Confocal Laser Scanning
Microscopy1
Christopher R. Shea,2 Margaret E. Sherwood, Thomas J. Flotte, Norah Chen, Manfred Scholz, and Tayyaba Hasan
Wellman Laboratories of Photomedicine, Department of Dermatology, Harvard Medical School, Massachusetts General Hospital, Boston, Massachusetts 02114
ABSTRACT
absorbs light efficiently at 514.5 nm (16), a wavelength whose
optical penetration into tissue is sufficient, and is even consid
Rhodamine 123 (R123) is a permeant, cationic, fluorescent dye that
ered optimal, for treatment of superficial malignancies such as
localizes preferentially within mitochondria of living carcinoma cells. carcinoma in situ of the urinary bladder (25). In vitro, R123
MGH-U1 human bladder carcinoma cells incubated in vitro with 10 ¿IM
phototoxicity causes significant inhibition of colony formation
R123 for 30 min and then irradiated at 514.5 nm with an argon ion laser
(16), proliferation (17), and uptake of tritiated thymidine (18)
underwent selective, phototoxic injury to mitochondria. Ultrastructurally,
of human bladder carcinoma cells at R123 concentrations and
treatment with R123 plus irradiation with 10 ,1/ciir caused selective,
radiant exposures that have no such effects when administered
progressive mitochondria! alterations consisting of disruption of cristae,
independently. The chemical and biological mechanisms of
vacuolization, swelling, increasing numbers of ring-shaped and angulated
mitochondria at 4 to 8 h after irradiation, and obliteration of many phototoxicity of R123 are unclear, including the primary pho
mitochondria at 24 to 48 h. Confocal laser scanning microscopy after
tochemistry responsible, the cellular lesions produced, and the
treatment with R123 plus irradiation with 10 to 30 J/air demonstrated
time course and functional consequences of photosensitized
altered uptake and localization of subsequently administered R123, ac
injury. In order to elucidate the details of the mechanisms of
companied by striking mitochondria! fragmentation. Irradiation caused a R123 phototoxicity, we have assessed the structural alterations
dose-dependent depletion of extractable R123, due to a photosensitized
of carcinoma cells by transmission EM and CLSM at various
efflux that began immediately and progressed by 4 h after irradiation
with 10 to 30 J/cm2; further uptake after reincubation in the presence of intervals after treatment in vitro with R123 followed by argon
ion laser irradiation at 514.5 nm. Morphological alterations
R123 was also quantitatively impaired in cells previously irradiated with
30 J/cm2.
have been correlated with functional injury to mitochondria, as
reflected by a reduced ability of irradiated cells to retain R123
and to concentrate it upon subsequent reincubation in its pres
INTRODUCTION
ence.
R1233 is the prototype of a group of permeant, cationic dyes
that have been widely investigated in recent years, both as
fluorescent probes and as potential agents for chemotherapy of
cancer. R123 preferentially localizes in undamaged mitochon
dria of living cells (1) largely because of electrophoretic forces
generated by the proton gradient across the mitochondrial inner
membrane (2). When mitochondria are injured and this gra
dient disturbed, R123 assumes a diffuse distribution in the
cytoplasm (3). Many types of carcinoma cells in vitro reportedly
have an increased avidity for R123 (4) because of an increased
electrical potential across the mitochondrial inner membrane
(2,5). Incubation in the presence of R123 at high concentrations
or for long periods causes mitochondrial toxicity (6-11), lead
ing to selective killing of certain carcinoma cells versus nontransformed epithelial cells in vitro (12). R123 chemotherapy
of cancer has been studied in vivo in rodent models (13, 14),
but toxic effects on normal organs limit its utility as monotherapy, even though there is a significantly greater uptake and
retention of R123 in experimental tumors than in normal
tissues (14, IS).
Combined treatment with R123 and visible-light irradiation
is phototoxic to cancer cells in vitro (16-23); photochemotherapy with low-dose R123 might therefore be an effective local
modality without severe systemic toxicity (24). R123 in cells
MATERIALS
AND METHODS
Cells. MGH-U1 cells (26), derived from a human transitional cell
carcinoma of the urinary bladder, were grown as subconfluent monolayers on glass coverslips in McCoy's Medium 5A with 25 ITIM4-(2hydroxyethyl)-l-piperazineethanesulfonic
acid buffer (Gibco Labora
tories, Grand Island, NY) supplemented with heat-inactivated 5% fetal
bovine serum (Gibco); incubation was at 37°Cin a humidified 95%
air:5% CO2 atmosphere. Cells in exponential growth were used for all
experiments. Routine cultures for Mycoplasma contamination were
consistently negative.
Radiation Source. The 514.5-nm emission from an argon ion laser
(Model Innova 100; Coherent, Inc., Palo Alto, CA) was directed to the
cell monolayer by a fiber optic system at an irradiance of 100 mW/cm2
as previously described (16). No detectable heating occurred at this
irradiance.
Photosensitization and Irradiation Protocol. Medium was aspirated
from cultures and replaced with 10 pM R123 (Eastman Kodak Co.,
Rochester, NY) in DPBS (Gibco) containing 0.49 mM MgCl2 H2O and
0.9 mM CaCl2 at pH 7.2. After incubation at 37°Cfor 30 min in the
dark, cultures were washed twice in R123-free DPBS, immediately
irradiated (3, 10, or 30 J/cm2) while in DPBS, and then either imme
diately fixed for EM, subjected to extraction in «-butylalcohol, or
covered with R123-free medium and incubated until ready for fixation,
extraction, or viewing by CLSM. Control experiments were performed
in parallel, with cultures exposed to R123-free DPBS with or without
Received 10/3/89; revised 2/2/90.
irradiation, or to R123 without irradiation. The radiant exposure range
The costs of publication of this article were defrayed in part by the payment
used has been shown previously to cause dose-dependent phototoxicity
of page charges. This article must therefore be hereby marked advertisement in
to MGH-U1 cells only after treatment with R123 (16-18).
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was sponsored by Office of Naval Research Contract NOOO14-86Electron Microscopy. Cells were washed twice in DPBS, fixed in 4%
K-0117; American Cancer Society Grant IN-173; NIH Grant GMA-1, 2 ROIglutaraldehyde, postfixed in 1% osmium tetroxide, dehydrated in a
AR25395; and Arthur O. and Gullan M. Wellman Foundation.
graded ethanol series, and embedded by inversion of Beem capsules
2To whom requests for reprints should be addressed.
containing Epon 812. Thin sections were stained with uranyl acetate
3The abbreviations used are: R123, rhodamine 123; CLSM, confocal laser
scanning microscopy; DOTC, doxycycline; DPBS. Dulbecco's phosphate-buffered
and lead citrate and viewed with an electron microscope (Model CM
saline; EM, electron microscopy; 'O2, singlet oxygen; TC, tetracycline.
10; Philips, Inc., Eindhoven, The Netherlands). Electron micrographs
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RHODAMINE 123 PHOTOTOXICITY
were taken of cultures fixed immediately and 1, 4, 8, 24, or 48 h after
treatment.
Confocal Laser Scanning Microscopy. At 1, 2, 4, or 26 h after
treatment, cells were incubated a second time in R123 solution (10 ^M
for 30 min), washed, mounted on glass slides with supplemental me
dium without phenol red, and then viewed by CLSM (Wild-Leitz, Inc.,
Wetzlar, Federal Republic of Germany) operated in the fluorescence
mode, with a xlOO, 1.32-numerical aperture, oil immersion objective,
using argon ion laser excitation at 488 nm and emission >515 nm.
Scanning over a smaller field size permitted higher magnification
images to be taken (zoom mode). Images were stored on an optical disc
and processed with a computer system equipped with image enhance
ment and coloring capabilities. Extended focus images were processed
from optical sections taken serially at z-plane increments of approxi
mately 0.5 litn (27). All CLSM experiments on any particular prepa
ration were completed within 15 min, to avoid artifactual changes in
cellular morphology and fluorescence. Preliminary experiments showed
that repeated scanning caused no detectable photobleaching or phototoxicity under the conditions used.
Dye Extraction and Quantification. Subconfluent cultures in plastic
Petri dishes were incubated with 10 MMR123 for 30 min, washed twice
in DPBS, covered with DPBS, irradiated (10 or 30 J/cm2) or kept in
the dark, treated briefly with 0.05% trypsin and 0.53 HIM EDTA
(Gibco), vigorously pipetted to obtain single cell suspensions, centrifuged, resuspended, counted (Model ZF; Coulter Electronics, Inc.,
Hialeah, FL), and immersed in fluorometric grade n-butyl alcohol.
Extraction was performed either immediately or 4 h after the irradiation
period; in additional experiments cells were reincubated with 10 ;<\i
R123 for 30 min at 4 h after the irradiation period and then immediately
subjected to the extraction procedures. Cellular R123 content was
calculated by comparison of the absorbance of extracts with standard
solutions using a diode array spectrophotometer (Model 8451 A; Hew
lett-Packard Co., Sunnydale, CA). Significance of differences in R123
content as a function of radiant exposure and time after irradiation was
assessed by nonparametric tests (Kruskal-Wallis for comparison of 3
groups and Whitney-Mann for paired comparisons); P values < 0.05
were considered statistically significant.
RESULTS
Electron Microscopy. Control cells (no R123, no irradiation)
had smooth, rounded plasma membranes with occasional, short
pseudopodia. Nuclei were eccentrically located and had in
dented contours and peripheral aggregation of chromatin. Organelles, especially Golgi apparatus and mitochondria, were
well developed and mainly located next to the nucleus. Most
mitochondria were elongated and had well-formed, evenly
spaced cristae (Fig. la). Cytoplasmic vacuoles, lipid droplets,
and myelin figures were rarely observed. Cells fixed immedi
ately or l h after irradiation with 10 J/cm2 alone, in the absence
of R123, appeared similar to control cells; those fixed from 4
to 48 h after irradiation showed slightly increased vacuolization
in the cytoplasm and slight disruption or loss of cristae. In
general, the alterations caused by treatment with R123 alone,
without irradiation, were subtle and did not vary with time after
treatment. Mitochondria of cells treated with R123 alone were
predominantly elongated (Fig. 1¿>);
some were rounded and
swollen or had focally disrupted cristae resulting in internal
vacuolization. Cells treated with R123 alone also contained a
greater number of cytoplasmic vacuoles, myelin figures, and
lipid droplets compared with control cells.
Combined treatment with R123 and irradiation with 10 J/
cm2 caused striking mitochondria! alterations, which pro
gressed over time. Immediately after irradiation no changes
were identified beyond the focal disruption of cristae seen in
cells treated with R123 alone (Fig. le). At l h after irradiation
some mitochondria appeared more distorted. At 4 h after
irradiation most mitochondria were moderately swollen, with
disrupted cristae (Fig. id); many mitochondria showed unusual
configurations, including ring-shaped and angulated forms with
variably thinned diameters. Other adjacent organdÃ-es were
entirely normal. At 8 h after irradiation there were more lipid
droplets and increased distortion of mitochondria with many
bizarre forms (Fig. \e). At 24 and 48 h after irradiation, many
mitochondria were obliterated (Fig. I/), and most of the re
maining mitochondria were swollen, had undergone severe
disruption or loss of cristae, and contained dense deposits in
the matrix. Cytoplasmic vacuoles (some containing amorphous
material) and myelin figures were prevalent, and there was
slight swelling of the Golgi apparatus. The nucleus appeared
normal.
Confocal Laser Scanning Microscopy. Living cells treated with
R123 alone, in the absence of laser irradiation, displayed bril
liant fluorescence exclusively within their mitochondria (Fig.
2); extramitochondrial sites (e.g., nucleus, cytoplasm) in unirradiated cells were free of R123 fluorescence. Serial optical
sectioning showed that the mitochondria comprised an inter
connecting system with multiple loops and branch points. In
typical optical sections, the apparent length of mitochondria
was about 5 ¡j.m.
By extended-focus processing of serial optical
sections, composite images were generated in which the overall
spatial continuity of mitochondria was apparent.
R123 phototoxicity caused striking CLSM findings (Fig. 3),
namely, an altered localization of R123 (variably within the
cytoplasm, nuclear membrane, and mitochondria) and struc
tural alterations of mitochondria themselves. Most mitochon
dria appeared fragmented and short (~1 ^m); some were swol
len, globular, or ring-shaped. In general the order of severity of
injury was 30 > 10 > 3 ~ 0 J/cm2, and 26 > 4 > 2 ~ 1 h, as
judged by qualitative morphological criteria. Phototoxic alter
ations were consistent over the course of multiple experiments,
with some regional variations in fluorescence intensity and
mitochondrial morphology. Irradiation caused no detectable
alterations of cells not pretreated with R123.
Iut rare-Millar R123 Content. Unirradiated cells showed a sig
nificant decrease in R123 content by 4 h after being covered
with R123-free medium (Fig. 4). Laser irradiation caused a
significantly greater decrease in R123 content, both immedi
ately after irradiation and at 4 h (P = 0.0001). The total R123
content also varied with radiant exposure in cells reincubated
with R123 at 4 h after the irradiation period (P = 0.0001).
After reincubation, however, unirradiated cells and cells previ
ously irradiated with 10 J/cm2 showed an identical net increase
in R123 content (mean change in content = 1.2 x 10~16mol/
cell); that is, net reuptake of R123 at this time was unaffected
by previous irradiation with 10 J/cm2. Cells previously irradi
ated with 30 J/cm2, on the other hand, showed both a signifi
cantly impaired reuptake of R123 (mean change in content =
7.2 x 10~17mol/cell, P < 0.02) and a low total R123 content
after reincubation with R123 (P = 0.0001), compared with
unirradiated cells.
DISCUSSION
R123 is a useful biological probe because of its high fluores
cence efficiency (28), relatively low toxicity when used at low
concentration for brief periods, and selective affinity for mito
chondria of living cells, in particular carcinoma cells. The
pattern and intensity of R123 fluorescence vary with the elec
trical potential gradients across the mitochondrial inner mem
brane and the plasma membrane (2), cell type (4), species (29),
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RHODAMINE
123 PHOTOTOXICITY
'«•«
:'•'
-,'"1 •
•'
Fig. 1. Transmission electron micros
copy demonstrates the ultrastructural ef
fects of R123-sensitized photochemical re
actions after irradiation at S14.S nm. In a,
control cells (no R123, no irradiation) ex
hibit elongated mitochondria with evenly
spaced cristae and a uniformly distributed,
electron-dense matrix. In b, cells fixed im
mediately after treatment for 30 min with
10 MMR123 alone (no irradiation) exhibit
well-formed mitochondria with minor in
ternal vacuolization (arrows). The number
of lipid droplets, myelin figures, and cytoplasmic vacuoles is increased, compared
with control cells. In c. cells pretreated
with 10 /¡MRI23 for 30 min and fixed
immediately after irradiation with 10 J/
cm2 exhibit mitochondria with some dis
ruption of cristae (arrows), similar to those
in cells treated with R123 alone (b). In d,
at 4 h after irradiation with 10 .I/cm2, cells
pretreated with 10 UM R123 for 30 min
show markedly swollen mitochondria with
disrupted cristae. Some mitochondria ex
hibit unusual configurations, predomi
nantly ring forms and angulated forms (ar
rows). In e, at 8 h after irradiation with 10
J/cm2, mitochondria of cells pretreated
with 10 MM R123 for 30 min show in
creased distortion. Very few normal ap
pearing mitochondria are present. There is
also increased lipid formation (asterisks).
In f, at 48 h after irradiation with 10 J/
cm2, most mitochondria in cells pretreated
with 10 MM for 30 min are obliterated.
Those mitochondria observed are swollen
and show almost total loss of cristae (arrows). Dense deposits are observed in the
mitochondria. Myelin figures are prevalent
(asterisks), and moderate swelling of the
Golgi apparatus is seen. (Bar = 1 ^m for
all electron micrographs.)
phase of the cell cycle (30), rate of proliferation (31), growth
and differentiation (32), and metabolic status and health of the
cell (33). For these reasons, R123 can be used as a probe of
both structure and function of mitochondria. The present study
exploits both this property of R123 and its ability to sensitize
phototoxic reactions within mitochondria selectively.
R123 treatment in the absence of irradiation reportedly can
alter mitochondrial ultrastructure (34, 35), but in our study the
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RHODAMINE
123 PHOTOTOXICITY
2 UJ
u
ce
LU
a
Ohr
4hr
4 hr, then
re-incubate with
R123
TIME POST IRRADIATION
Fig. 4. Intracellular R123 content as a function of radiant exposure. Cells
were pretreated with R123 (10 ^M for 30 min) and either shielded from light or
irradiated at 514.5 nm. D, unirradiated cultures;E3, cultures irradiated with 10 J/
cm2; •.cultures irradiated with 30 J/cm2. There is a significant dose-dependent
and time-dependent decrease in R123 content, and the R123 content of cells
reincubated with R123 at 4 h after irradiation also varies with dose (radiant
exposure). Columns, mean of 9 to 18 cultures at each entry; bars, SE.
Fig. 2. High-magnification (zoom 4) CLSM of a perinuclear region of a single,
unfixed cell treated with 10 I¿M
R123 for 30 min without irradiation. Extended
focus composite image taken from 10 single scans demonstrates the complex,
largely continuous mitochondria! system. The intensity of fluorescence is propor
tional to color (white > yellow > red > black). Note that the nucleus (in the lower
left corner) and intermitochondrial cytoplasmic spaces do not fluoresce. (Bar = 5
(im).
Fig. 3. Normal magnification, extended focus CLSM of unfixed cells 4 h after
R123 pretreatment (10 ^M for 30 min) and irradiation with 10 J/cm2 at 514.5
nm. Mitochondria are markedly fragmented but tend to remain brightly fluores
cent. A background of diffuse fluorescence, including localization in the nuclear
membrane, is apparent. (Bar =
changes induced by R 123 alone were rather subtle; this finding
is consistent with the lack, in our system, of any detectable
effects of R 123 alone on colony formation, thymidine incor
poration, proliferation, or vital staining (16-18), probably be
cause of the relatively low intracellular R 123 concentration
attained under these incubation conditions. Induction of more
striking alterations by treatment with R123 alone generally
requires higher ambient R123 concentrations or longer incu
bation times, which lead to higher intracellular R123 content
and increased toxicity (34). LI210 cells treated with sufficient
R123 in the absence of irradiation reportedly show intracristal
vacuolization and distortion, an increase in the number of
matrix granules, and ring-shaped mitochondria (35). In the
present study, very similar findings were prominent in MGHUl cells treated with low-dose R123 plus 10 J/cm2 laser light.
Laser irradiation thus appears to cause a true potentiation of
the intrinsic toxicity of R123, even inducing similar ultrastruc
tural alterations. Ring-shaped mitochondria (which are rarely
seen in normal cells) are probably a consequence of mitochon
dria! fragmentation; they can be induced by other mitochondrial
toxins as well (36).
The ultrastructural changes induced in mitochondria by R123
phototoxicity differ greatly from those seen in cells treated with
the phototoxic drug DOTC and then irradiated with ultraviolet
A, 320-400 nm (37); the differences are notable because DOTC,
like other TCs (38), is also concentrated selectively within
mitochondria and sensitizes mitochondrion-specific phototox
icity. DOTC phototoxicity causes mitochondria to swell mark
edly (up to 5 times their normal diameter) and to lose the ability
to concentrate R123 (37, 39); these responses begin by 10 min
after irradiation, are maximal by 1 h, and are partially reversed
by 4 h. In contrast, R123 phototoxicity causes mitochondrial
alterations that are most evident many hours after irradiation
and are not reversed by 48 h. Furthermore, the phototoxic
swelling induced with R123 is not as massive as with DOTC;
rather, R123 phototoxicity primarily causes the mitochondria
to undergo fragmentation and bizarre changes in configuration.
These different results probably reflect differences in both pri
mary photochemistry responsible for injury and the specific
suborganelle site of injury. TC phototoxicity is largely oxygendependent (40), and the generation of 'O2 is a major phototoxic
mechanism (39-41). 'O2 efficiently induces membrane oxida
tion (42), leading to increased permeability and swelling, gen
erally within a few minutes after irradiation (43). In contrast,
R123 phototoxicity is progressively manifested at much later
times after irradiation, consistent with the low (<1%) quantum
yield of 'O2 from R123 photoreactions (16) due to the low
efficiency of intersystem crossing to the triplet manifold (28,
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RHODAMINE
123 PHOTOTOXICITY
44). The biochemical lesion(s) produced by R123 photosensitization must be investigated further; one plausible target is the
F0F, ATPase of the mitochondria! inner membrane, a major
site of action of R123 in the absence of irradiation (5).
The high spatial resolution of CLSM images is a consequence
of the ability to perform optical sectioning through the z-plane,
by suppression of out-of-focus optical planes (27, 45). In our
study, continuity and interconnections of the unirradiated
MGH-U1 cell mitochondria are apparent, a form of mitochondrial organization seen in only a few cell types such as mouse
3T6 cells (1). In contrast, the MGH-U1 cell mitochondria are
fragmented after R123 photosensitization; furthermore, R123
is present in the nuclear membrane of irradiated cells only,
reflecting a redistribution in response to mitochondrial injury.
Photobleaching of R123 with CLSM in this study is less than
with conventional epifluorescence microscopy, because the in
stant storage of images obviates the need for prolonged illumi
nation during viewing or photography. Thus the images ob
tained are stable, and no correction needs to be made for
dynamic changes such as fluorescence redistribution in this
study.
The uptake and efflux of R123, demonstrated qualitatively
by CLSM, have been quantified by extraction. At 4 h after
being covered with R123-free medium, unirradiated cells have
lost about half the R123 initially present (Fig. 4); the rate of
efflux is very similar to that demonstrated in other carcinoma
cell lines (46). After a 30-min reincubation in 10 fiM R123
solution, unirradiated MGH-U1 cells take up the same incre
mental amount of R123 as they did after the original incubation,
indicating that mitochondrial binding sites of R123 (47) are
not saturated; in fact, R123 remains selectively localized to
mitochondria of unirradiated cells even at a total content >1 x
10~15mol/cell (achieved by incubating cells for 60 min in 100
fiM R123 solution).4
Laser irradiation of cells markedly alters their R123 content.
Within minutes after irradiation with 10 J/cm2 and 30 J/cm2,
the mean R123 content drops to approximately 50% and 27%,
respectively, of that of unirradiated cells. This immediate pho
tosensitized efflux is easily detected even though injury is not
yet apparent by EM; thus R123 content is a very sensitive
measure of functional injury to mitochondria. Photodegrada
tion of R123 probably contributes little to this immediate
decrease, for only 10% photodegradation occurs in R123 solu
tion irradiated with 40 J/cm2 (17). R123 continues to efflux
from cells by 4 h after irradiation, at a rate directly proportional
to the radiant exposure (Fig. 2); conversely, the total R123
content of cells incubated a second time in R123 solution after
4 h is inversely related to the radiant exposure. Paradoxically,
cells previously treated with R123 plus 10 J/cm2 irradiation
take up a normal incremental amount of R123 after 4 h. CLSM
shows, however, that the R123 is abnormally distributed, par
tially within altered mitochondria and partially within the cytoplasmic compartment and nuclear membrane. Thus, bulk
uptake of R123 cannot be the sole criterion of mitochondrial
injury but must be correlated with other morphological and
functional data. [For example, we have reported elsewhere that
R123 pretreatment plus irradiation with 10 J/cm2 causes almost
total inhibition of cellular proliferation for the first 48 h after
irradiation (17).] Compared with unirradiated cells, cells irra
diated with 30 J/cm2 take up a significantly lower incremental
amount of R123 upon reincubation; CLSM shows that this
R123 is localized mainly in the cytoplasm, and therefore its
* C. R. Shea et al., unpublished data.
uptake presumably results mainly from the electrical potential
across the plasma membrane, which appears intact even at this
rather high radiant exposure (16). The altered localization of
R123 after laser irradiation may be a consequence of nonspe
cific impairment of the mitochondrial inner membrane poten
tial or perhaps photosensitized alteration of specific binding
sites of R123.
In summary, R123 photosensitization causes injury selec
tively to mitochondria of cultured carcinoma cells, because of
toxic photochemical reactions at its site of preferential localiza
tion. This organelle-specific phototoxic injury, with its distinc
tive morphological and functional consequences, is of interest
for basic studies of photobiology and bioenergetics and may
lead to the rational synthesis of new photosensitizers and to
further use of tumor mitochondria as targets of novel forms of
photochemotherapy.
ACKNOWLEDGMENTS
We thank Dr. Chi-Wei Lin for his gift of MGH-U1 cells, Dr. Reiner
Peters for help in the CLSM studies, Dr. Sewon Kang for statistical
consultation, Dr. Victor Tron and Dr. Randy Byers for helpful discus
sions and criticisms, Wild-Leitz for the loan of CLSM equipment, and
Gabriele Vogt for expert secretarial help.
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Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1990 American Association for Cancer Research.
Rhodamine 123 Phototoxicity in Laser-irradiated MGH-U1
Human Carcinoma Cells Studied in Vitro by Electron
Microscopy and Confocal Laser Scanning Microscopy
Christopher R. Shea, Margaret E. Sherwood, Thomas J. Flotte, et al.
Cancer Res 1990;50:4167-4172.
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